The search for strategies aimed at preserving the effectiveness of currently available antibiotics. Our model system is the aminoglycoside 6'-N-acetyltransferase type Ib [AAC (6')-Ib], an enzyme that mediates resistance to amikacin and other clinically relevant aminoglycosides. These antibiotics, alone or in combination with ?- lactams or vancomycin, are commonly used for the treatment of serious infections caused by several gram- negative pathogens as well as by some gram-positives. The long term goal of this research is to develop pharmacological tools to selectively inhibit the expression of aac(6')-Ib and the AAC(6')-Ib enzyme to achieve phenotypic conversion to susceptibility. In the previous granting period we have identified oligoribonucleotide sequences, known as external guide sequences (EGSs), that elicit RNase P-mediated cleavage of aac(6')-Ib mRNA in vitro and reduce the levels amikacin resistance in vivo. However, since a common problem of antisense strategies is that the inhibitory activity is not potent enough for an effective antimicrobial effect, specific aim 1 will consist of testing the enhancing effect of mixes of EGSs targeting different regions of aac(6')-Ib mRNA or mixes of EGSs that target aac(6')-Ib mRNA and an mRNA encoded by an essential gene such as ftsZ. While achieving this specific aim will be an important step towards developing antisense compounds to preserve the efficacy of amikacin and other aminoglycosides, many challenges will remain to be solved. Some of these problems are: a) any successful utilization of antisense compounds must utilize nuclease resistant oligonucleotide analogs that can elicit RNase P-mediated cleavage of the target mRNA, b) methodologies that help deliver the antisense compounds inside the cytosol are very limited, and c) since aac(6')-Ib, as it is the case with many resistance genes, is often located in high copy number plasmids it may be very difficult to completely turn off expression. Specific aims 2 - 4 have been designed to search for solutions to these problems. In specific aim 2 we propose to perform a systematic analysis of the ability of different nuclease resistant oligonucleotide analogs to identify those most appropriate to induce RNAse P- mediated cleavage of the target mRNA. In specific aim 3 we propose to test two methodologies to deliver the antisense compounds inside the cell's cytosol. One of them consists of encapsulating the oligomers into liposomes using a large variety of formulations including lipids and proteins, and the other consists of conjugating the oligomers to permeabilizer peptides. Specific aim 4 has been designed to solve the problem of residual expression of the resistance gene. We will search for peptides or small molecules that act as enzymatic inhibitors. For this we will apply the positional scanning strategy using synthetic peptide and small molecule combinatorial libraries. PUBLIC HEALTH RELEVANCE: Aminoglycosides are widely used antibiotics for treatment of serious bacterial infections but unfortunately the success rate of treatments with these antibiotics has gone down due to the appearance of multiresistant strains. An option to deal with the problem of drug resistance is to develop strategies to prolong the effectiveness of currently available antibiotics. This project will contribute to extend the useful life of clinically important aminogylcosides.